This proposal capitalizes on recent methodological advances in protein engineering and structural analysis in order to further understanding of the relationship between the structure and function of the antibody combining site. Digoxin specific antibodies are a model system in which antibody and hapten structures are designed and modified to affect biologic activity through alterations in binding specificity. Anti- digoxin antibodies are proven therapeutic agents for diagnosis and reversal of toxicity due to digitalis glycosides. Monoclonal antidigoxin antibodies display exceptional affinity for their site- filling hydrophobic ligand, for which there are hundreds of structurally defined congeners of known stereochemistry. The now proven feasibility of cloning of antibody variable region genes, in vitro mutagenesis, and expression of antibodies or antibody binding fragments following transfection in eukaryotes, or in prokaryotes (E coli) permits engineering of mutants providing direct tests of the "rules" for antibody complementarity. These studies are complemented and made feasible by analyses of tertiary structure employing X-ray crystallography, nuclear magnetic resonance spectroscopy, and computer modeling. Proposed experiments include: 1) structural and binding analysis of spontaneous variable region somatic mutants with altered binding selected by cell sorting obtained from secondary response hybridomas. 2) The recently determined crystal structures of the Fab:digoxin complex of the cognate antibody 26-10 and a nonbinding mutant serve as the basis for engineering of new variable region mutants with altered binding. The design of mutants is based also on results of NMR studies of Fv, predictive computer modeling, and studies of spontaneous mutants in hand. 3) Engineer by in vitro mutagenesis binding variants of antibody 40-50 for which crystallographic refinement is near completion. These studies parallel those for 26-10, which has different primary structure and binding specificity, providing a unique opportunity to compare antibodies to the same hapten. 4) Determine relative chain contribution to binding in antibody sets sharing VL regions. 5) Fv, single chain Fv and Fab fragments of monoclonal antibodies and mutants are expressed and their binding characterized, in conjunction with NMR and crystallographic studies. The manipulation of antibody genes and proteins for the elucidation of the structural basis of antigen-antibody complementarity is a paradigm important in the wider application of antibodies as therapeutic tools, whether used directly in immunotherapy as Fv or Fab fragments, or targeted by linkage to effector molecules.